RESUMEN
Human skin is constantly exposed to solar ultraviolet radiation (UVR), the most prevalent environmental carcinogen. Humans have the unique ability among mammals to respond to UVR by increasing their skin pigmentation, a protective process driven by melanin synthesis in epidermal melanocytes. The molecular mechanisms used by melanocytes to detect and respond to long-wavelength UVR (UVA) are not well understood. We recently identified a UVA phototransduction pathway in melanocytes that is mediated by G protein-coupled receptors and leads to rapid calcium mobilization. Here we report that in human epidermal melanocytes physiological doses of UVR activate a retinal-dependent current mediated by transient receptor potential A1 (TRPA1) ion channels. The TRPA1 photocurrent is UVA-specific and requires G protein and phospholipase C signaling, thus contributing to UVA-induced calcium responses to mediate downstream cellular effects and providing evidence for TRPA1 function in mammalian phototransduction. Remarkably, TRPA1 activation is required for the UVR-induced and retinal-dependent early increase in cellular melanin. Our results show that TRPA1 is essential for a unique extraocular phototransduction pathway in human melanocytes that is activated by physiological doses of UVR and results in early melanin synthesis.
Asunto(s)
Canales de Calcio/metabolismo , Canales de Calcio/efectos de la radiación , Fototransducción/fisiología , Fototransducción/efectos de la radiación , Melanocitos/metabolismo , Melanocitos/efectos de la radiación , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/efectos de la radiación , Canales de Potencial de Receptor Transitorio/metabolismo , Canales de Potencial de Receptor Transitorio/efectos de la radiación , Animales , Células CHO , Canales de Calcio/genética , Señalización del Calcio/efectos de la radiación , Células Cultivadas , Cricetinae , Cricetulus , Proteínas de Unión al GTP/metabolismo , Humanos , Melaninas/biosíntesis , Modelos Biológicos , Proteínas del Tejido Nervioso/genética , Técnicas de Placa-Clamp , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efectos de la radiación , Transducción de Señal/efectos de la radiación , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/genética , Rayos UltravioletaRESUMEN
In vertebrate rods, photoisomerization of the 11-cis retinal chromophore of rhodopsin to the all-trans conformation initiates a biochemical cascade that closes cGMP-gated channels and hyperpolarizes the cell. All-trans retinal is reduced to retinol and then removed to the pigment epithelium. The pigment epithelium supplies fresh 11-cis retinal to regenerate rhodopsin. The recent discovery that tens of nanomolar retinal inhibits cloned cGMP-gated channels at low [cGMP] raised the question of whether retinoid traffic across the plasma membrane of the rod might participate in the signaling of light. Native channels in excised patches from rods were very sensitive to retinoid inhibition. Perfusion of intact rods with exogenous 9- or 11-cis retinal closed cGMP-gated channels but required higher than expected concentrations. Channels reopened after perfusing the rod with cellular retinoid binding protein II. PDE activity, flash response kinetics, and relative sensitivity were unchanged, ruling out pharmacological activation of the phototransduction cascade. Bleaching of rhodopsin to create all-trans retinal and retinol inside the rod did not produce any measurable channel inhibition. Exposure of a bleached rod to 9- or 11-cis retinal did not elicit channel inhibition during the period of rhodopsin regeneration. Microspectrophotometric measurements showed that exogenous 9- or 11-cis retinal rapidly cross the plasma membrane of bleached rods and regenerate their rhodopsin. Although dark-adapted rods could also take up large quantities of 9-cis retinal, which they converted to retinol, the time course was slow. Apparently cGMP-gated channels in intact rods are protected from the inhibitory effects of retinoids that cross the plasma membrane by a large-capacity buffer. Opsin, with its chromophore binding pocket occupied (rhodopsin) or vacant, may be an important component. Exceptionally high retinoid levels, e.g., associated with some retinal degenerations, could overcome the buffer, however, and impair sensitivity or delay the recovery after exposure to bright light.
Asunto(s)
Canales Iónicos/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Retinoides/farmacología , 1-Metil-3-Isobutilxantina/farmacología , 3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Ambystoma , Animales , GMP Cíclico/biosíntesis , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Diterpenos , Guanilato Ciclasa/metabolismo , Canales Iónicos/antagonistas & inhibidores , Luz , Microespectrofotometría , Técnicas de Placa-Clamp , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/efectos de la radiación , Retinaldehído/metabolismo , Retinaldehído/farmacología , Retinoides/metabolismo , Proteínas de Unión al Retinol/farmacología , Proteínas Plasmáticas de Unión al Retinol , Rodopsina/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Vitamina A/farmacologíaRESUMEN
Cyclic nucleotide-gated (CNG) channels have been well characterized in the sensory receptors of vision and olfaction, but their characteristics in other tissues remain largely unknown. Here, we report characterization of a novel brain-specific CNG channel from zebrafish. Unique among CNG channels, the transcript is expressed mainly in the brain. When expressed in Xenopus oocytes, the channel's electrophysiological properties are distinct compared to CNG channels from either rods (CNGA1), olfactory receptors (CNGA2), or cones (CNGA3). The channel is less sensitive to cAMP than cGMP (K(1/2) of 280 and 7 microM, respectively), with a maximum cAMP efficacy at least 80% of that with saturating levels of cGMP. The single-channel conductance of 58pS is larger than most other CNG channels. Like other CNG channels the channel is relatively nonselective among monovalent cations. However, unlike other CNG channels, there was rundown of the macroscopic current within 30-100 min after patch excision.
Asunto(s)
Química Encefálica , Canales Iónicos/fisiología , Proteínas de Pez Cebra/fisiología , Secuencia de Aminoácidos , Animales , AMP Cíclico/farmacología , GMP Cíclico/farmacología , Canales Iónicos/química , Canales Iónicos/efectos de los fármacos , Datos de Secuencia Molecular , Alineación de Secuencia , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/efectos de los fármacosAsunto(s)
AMP Cíclico/metabolismo , Plasticidad Neuronal/fisiología , Neuronas Receptoras Olfatorias/fisiología , Animales , Calcio/metabolismo , Canales de Cloruro/fisiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1 , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Hidrólisis , Canales Iónicos/fisiología , Ratones , Hidrolasas Diéster Fosfóricas/metabolismoRESUMEN
PURPOSE: To determine whether inhibition of cyclic nucleotide-gated (CNG) ion channels by retinoids might be useful in treating degenerative retinal diseases in which either the CNG channels are hypersensitive to 3',5'-cyclic guanosine monophosphate (cGMP) or the photoreceptor cGMP concentration is elevated. METHODS: Patch clamp (electrophysiological) methods were used to measure activation by cGMP of wild-type human cone (hCNGA3), mutant cone (hCNGA3-N471S), and wild-type bovine rod (bCNGA1) CNG channels heterologously expressed in Xenopus oocytes. Cyclic GMP-activated currents were measured in excised, inside-out membrane patches before and after treatment with either all-trans retinal (ATR) or all-trans C22 aldehyde, which is too long to fit into the chromophore binding pocket of opsin and therefore cannot activate the visual transduction cascade. RESULTS: At physiological cGMP concentrations, 150 nM ATR reduced the open probability of the mutant cone CNG channel by reducing its apparent cGMP affinity to that of the normal cone channel. Furthermore, all-trans C22 aldehyde similarly inhibited the mutant cone channel as well as normal rod and cone CNG channels. CONCLUSIONS: Our results raise the possibility that retinoids, such as all-trans C22 aldehyde, that inhibit CNG channels without affecting the transduction cascade, may be useful in treating degenerative retinal diseases in which either the cGMP concentration is elevated or the CNG channels are hypersensitive to cGMP.
Asunto(s)
GMP Cíclico/farmacología , Canales Iónicos/genética , Canales Iónicos/metabolismo , Mutación , Retinitis Pigmentosa/genética , Retinoides/farmacología , Animales , Asparagina , Bovinos , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Humanos , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/efectos de los fármacos , Oocitos , Técnicas de Placa-Clamp , Retinitis Pigmentosa/tratamiento farmacológico , Serina , Transducción Genética , Vitamina A/farmacología , XenopusRESUMEN
A molecular assembly consisting of a redox enzyme, NADH peroxidase, a metallized double-helical peptide, and a gold nanoparticle immobilized onto a gold wire derivatized with a benzenedithiol compound, initiated and conducted redox signals in the presence of H(2)O(2) and NADH. The current generated by the binding of NADH, the electron donor, was transduced through the molecular assembly with apparently little loss of signal to the solution. The currents measured correlate to an electron transfer rate constant on the order of 3,000 s(-1) within each assembly. This electron transfer rate is two orders of magnitude higher than the endogenous electron transfer rate from NADH to the native enzyme, 27 s(-1). This rate indicates that the metallized peptide is in a conformation conducive for electron transfer and, in conjunction with the redox enzyme, can form effective conduits of electrical signals. This work demonstrates the feasibility of utilizing designed and highly efficient biomolecular assemblies for the production of ultra-sensitive, in-situ biosensors.
Asunto(s)
Técnicas Biosensibles/instrumentación , Oro/química , Peróxido de Hidrógeno/análisis , Microelectrodos , Nanotecnología/instrumentación , Péptidos/química , Peroxidasas/química , Técnicas Biosensibles/métodos , Instalación Eléctrica , Electroquímica/instrumentación , Electroquímica/métodos , Enzimas Inmovilizadas/química , Diseño de Equipo , Análisis de Falla de Equipo , Peróxido de Hidrógeno/química , Metales/química , Nanoestructuras/análisis , Nanoestructuras/química , Nanotecnología/métodos , Oxidación-ReducciónRESUMEN
Rod vision is initiated when 11-cis-retinal, bound within rhodopsin, absorbs a photon and isomerizes to all-trans-retinal (ATR). This triggers an enzyme cascade that lowers cGMP, thereby closing cyclic nucleotide-gated (CNG) channels. ATR then dissociates from rhodopsin, with bright light releasing millimolar levels of ATR. We have recently shown that ATR is a potent closed-state inhibitor of the rod CNG channel, and that it requires access to the cytosolic face of the channel (McCabe, S.L., D.M. Pelosi, M. Tetreault, A. Miri, W. Nguitragool, P. Kovithvathanaphong, R. Mahajan, and A.L. Zimmerman. 2004. J. Gen. Physiol. 123:521-531). However, the details of the interaction between the channel and ATR have not been resolved. Here, we explore the nature of this interaction by taking advantage of specific retinoids and retinoid analogues, namely, beta-ionone, all-trans-C15 aldehyde, all-trans-C17 aldehyde, all-trans-C22 aldehyde, all-trans-retinol, all-trans-retinoic acid, and all-trans-retinylidene-n-butylamine. These retinoids differ in polyene chain length, chemical functionality, and charge. Results obtained from patch clamp and NMR studies have allowed us to better define the characteristics of the site of retinoid-channel interaction. We propose that the cytoplasmic face of the channel contains a retinoid binding site. This binding site likely contains a hydrophobic region that allows the ionone ring and polyene tail to sit in an optimal position to promote interaction of the terminal functional group with residues approximately 15 A away from the ionone ring. Based on our functional data with retinoids possessing either a positive or a negative charge, we speculate that these amino acid residues may be polar and/or aromatic.
Asunto(s)
Sitios de Unión/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Retinoides/farmacología , Animales , Sitios de Unión/fisiología , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Relación Dosis-Respuesta a Droga , Electrofisiología , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/fisiología , Norisoprenoides/química , Norisoprenoides/metabolismo , Oocitos , Técnicas de Placa-Clamp , Células Fotorreceptoras Retinianas Bastones/fisiología , Retinaldehído/metabolismo , Retinaldehído/farmacología , Retinoides/química , Retinoides/metabolismo , Xenopus laevis/genética , Xenopus laevis/fisiologíaAsunto(s)
Activación del Canal Iónico/fisiología , Canales Iónicos/química , Canales Iónicos/fisiología , Sales (Química)/metabolismo , Animales , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Canales de Potasio , Unión Proteica , Conformación Proteica , Sales (Química)/química , Relación Estructura-ActividadRESUMEN
Rod vision begins when 11-cis-retinal absorbs a photon and isomerizes to all-trans-retinal (ATR) within the photopigment, rhodopsin. Photoactivated rhodopsin triggers an enzyme cascade that lowers the concentration of cGMP, thereby closing cyclic nucleotide-gated (CNG) ion channels. After isomerization, ATR dissociates from rhodopsin, and after a bright light, this release is expected to produce a large surge of ATR near the CNG channels. Using excised patches from Xenopus oocytes, we recently showed that ATR shuts down cloned rod CNG channels, and that this inhibition occurs in the nanomolar range (aqueous concentration) at near-physiological concentrations of cGMP. Here we further characterize the ATR effect and present mechanistic information. ATR was found to decrease the apparent cGMP affinity, as well as the maximum current at saturating cGMP. When ATR was applied to outside-out patches, inhibition was much slower and less effective than when it was applied to inside-out patches, suggesting that ATR requires access to the intracellular surface of the channel or membrane. The apparent ATR affinity and maximal inhibition of heteromeric (CNGA1/CNGB1) channels was similar to that of homomeric (CNGA1) channels. Single-channel and multichannel data suggest that channel inhibition by ATR is reversible. Inhibition by ATR was not voltage dependent, and the form of its dose-response relation suggested multiple ATR molecules interacting per channel. Modeling of the data obtained with cAMP and cGMP suggests that ATR acts by interfering with the allosteric opening transition of the channel and that it prefers closed, unliganded channels. It remains to be determined whether ATR acts directly on the channel protein or instead alters channel-bilayer interactions.
Asunto(s)
GMP Cíclico/metabolismo , Activación del Canal Iónico/fisiología , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/fisiología , Modelos Biológicos , Células Fotorreceptoras Retinianas Bastones/fisiología , Vitamina A/farmacología , Animales , Bovinos , Simulación por Computador , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Relación Dosis-Respuesta a Droga , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Oocitos/fisiología , Xenopus laevisRESUMEN
Members of the voltage-gated family of ion channels generally demonstrate rotational symmetry about their pore regions. Recent evidence suggests that a subset of this family, the cyclic nucleotide-gated channels, may deviate from this pattern of rotational symmetry by having 3A:1B subunit stoichiometry. This finding raises many questions about the function, assembly, and trafficking of these and related ion channels and about the functional nonequivalence of subunits with identical amino acid sequences.
Asunto(s)
Canales Iónicos/fisiología , Sistema Nervioso/metabolismo , Neuronas/metabolismo , Animales , Señalización del Calcio/fisiología , Proteínas Portadoras/fisiología , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Humanos , Estructura Terciaria de Proteína/fisiología , RotaciónRESUMEN
In retinal rods, light-induced isomerization of 11-cis-retinal to all-trans-retinal within rhodopsin triggers an enzyme cascade that lowers the concentration of cGMP. Consequently, cyclic nucleotide-gated (CNG) ion channels close, generating the first electrical response to light. After isomerization, all-trans-retinal dissociates from rhodopsin. We now show that all-trans-retinal directly and markedly inhibits cloned rod CNG channels in excised patches. 11-cis-retinal and all-trans-retinol also inhibited the channels, but at somewhat higher concentrations. Single-channel analysis suggests that all-trans-retinal reduces average open probability of rod CNG channels by inactivating channels for seconds at a time. At physiological cGMP levels, all-trans-retinal inhibited in the nanomolar range. Our results suggest that all-trans-retinal may be a potent regulator of the channel in rods during the response to bright light, when there is a large surge in the concentration of all-trans-retinal.